Infection with hepatitis C virus (HCV) has a major medical impact worldwide leading to chronic infections, cirrhosis of the liver and cancer (Di Bisceglie, A. M., and Bacon, B. R. (1999) Scientific American (Oct.): 80-85). Worldwide over 100 million people are chronically infected (Alter, M. J. (1997) Hepatology, 26(suppl. 1): 62S-65S; Hoofnagle, J. H., and DiBisceglie, A. M. (1997) New Engl. J. Med., 336: 337-356). In the U.S. almost 4 million people are chronically infected, and almost 9,000 people die annually from the disease (Chisari, F. V., and Ferrari, C. (1997) Viral hepatitis. In: Viral Pathogenesis. Ed: Nathanson, N., et al. Lippincott-Raven Publishers, Philadelphia, 1997: 745-778). The chimpanzee (Pan troglodites) is the only animal model with which to study the pathogenesis of hepatitis C virus (HCV) infection of humans (Houghton, M. Hepatitis C Viruses. In: Fields Virology, third edition. Eds; Fields, B. H., Knipe, D. M., Howley, P. M., et al. Lippencott-Raven Publishers, Philadelphia, 1996: 1035-1058).
HCV is a member of the viral Family Flaviviridae which are viruses that contain a positive sense, single-stranded RNA genome within an enveloped core (Miller, R. H., and Purcell, R. H. (1990) Proc. Natl. Acad. Sci. USA, 87: 2057-2061). Related members of this viral family which use similar genomic organization and replication strategies are the flaviviruses, pestiviruses, and hepatitis C viruses (Rice, C. M. Flaviviridae: The Viruses and Their replication. In: Fields Virology, third edition. Fields, B. H., Knipe, D. M., and Howley, P. M., et al, eds. Lippencott-Raven Publishers, Philadelphia, 1996: 931-959). A related pestivirus is bovine viral diarrhea virus (BVDV) which is infectious to cattle and can lead to persistent infections. Pestivirus diseases are widespread and still of economic importance to the livestock industry (Houe, H. (1999) Veterinary Microbiology, 64: 89-107).
Bovine viral diarrhea virus (BVDV), as first described by Baker et al, was transmitted to and serially passaged in rabbits (Baker, J. A. et al. (1954) Am. J. Vet. Res., Oct.1954: 525-531). Further investigation led to the adaptation of BVDV to rabbits and selecting a ‘biotype’ which was less cytopathic (Fernelius, A. L. et al. (1969) Am. J. Vet. Res., 30 (9): 1541-1550). These early studies demonstrated transmission of a viral pathogen, considered restricted to ruminant species, to a more convenient laboratory animal model.
The woodchuck infected with the woodchuck hepatitis virus (WHV) is an art recognized and accepted model for investigation of the pathogenesis of hepatitis B virus (HBV) infections of humans (Menna, S., and Tennant, B. C. (1999) Nature Med., 5(10): 1125-1126). This animal model has been instrumental in the development of antiviral treatments for chronic hepadnaviral (WHV, HBV) infections (Korba, B. E., et al. Woodchuck Virus Infection as a Model for the Development of Antiviral Therapies Against HBV. In: Viral Hepatitis and Liver Disease. Eds: Hollinger, F. B., Lemon, S. M., and Margolis, H. S., Williams and Wilkens, Baltimore, 1991: 556-559). Additionally, the human delta hepatitis virus has been adapted to infection of woodchucks as an alternative model to the chimpanzee (Taylor, J. et al. (1987) J. Virol., 61: 2981-2985; Ponzetto, A. et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 2208-2212).
A significant focus of current antiviral research is directed to the development of improved methods of treatment of chronic HCV infections in humans (Di Besceglie, A. M. and Bacon, B. R., Scientific American, Oct.: 80-85, (1999). Discovery of new treatments for the treatment of HCV has been hindered by the lack of suitable animal models. Currently, the only antiviral drugs available toward the treatment of chronic HCV infection in humans are alpha-interferon and ribavirin (Hoofnagle, J. H., and DiBisceglie, A. M. (1997) New Engl. J. Med., 336: 337-356).
BVDV is often used as a model for the development of HCV surrogate molecular target assays, because of the difficulties of growing HCV in vitro. BVDV and HCV have a high degree of genetic homology, common replication strategies and it is believed the same sub-cellular location for viral envelopment (Collet, M. S. (1992) Comp. Immun. Micro. Infect. Dis. 15, 145-155).
The nucleotide sequence of the RNA genome of the human hepatitis C virus (HCV) has been determined from overlapping cDNA clones. The sequence (9379 nucleotides) has a single large open reading frame that could encode a viral polyprotein precursor of 3011 amino acids. While there is little overall amino acid and nucleotide sequence homology with other viruses, the 5′ HCV nucleotide sequence upstream of this large open reading frame has substantial similarity to the 5′-termini of pestiviral genomes, in particular BVDV. A portion of the 5′-UTR (a 332-341 nucleotide region) contains a sequence with a 47% sequence homology to bovine viral diarrhoea virus (BVDV).
The polyprotein from the open reading frame of HCV also has significant sequence similarity to the NTP-binding helicases (approximately between amino acids 1230 and 1500) encoded by animal pestiviruses, and it contains sequence motifs widely conserved among viral-encoded RNA-dependent RNA polymerase (between amino acids 2703-2739, containing six highly conserved residues) and trypsin-like proteases. A basic, presumed nucleocapsid domain is located at the N terminus upstream of a region containing numerous potential N-linked glycosylation sites. These HCV domains are located in the same relative position as observed in the pestiviruses and flaviviruses and the hydrophobic profiles of all three viral polyproteins are similar. (Choo, et al. “Genetic organization and diversity of the hepatitis C virus” 1991, Proc. Natl. Acad. Sci. 88, 2451-2455).
In addition, BVDV and HCV both have an NS4A cofactor requirement for the NS3 serine protease; and BVDV exhibits polyprotein cleavages similar to that seen in HCV, which occur in the nonstructural region (NS5 A/B).
Treatment of HCV Infection with Ribavirin
Ribavirin (1-β-D-ribofuranosyl-1-1,2,4-triazole-3-carboxamide) is a synthetic, non-interferon-inducing, broad spectrum antiviral nucleoside analog sold under the trade name Virazole™ (The Merck Index, 11th edition, Editor: Budavari, S., Merck & Co., Inc., Rahway, N.J., p1304, 1989). U.S. Pat. Nos. 3,798,209 and RE29,835 disclose and claim ribavirin.
Ribavirin is structurally similar to guanosine, and has in vitro activity against several DNA and RNA viruses including Flaviviridae (Davis. Gastroenterology 118:S104-S114, 2000). Ribavirin reduces serum amino transferase levels to normal in 40% of patients, but it does not lower serum levels of HCV-RNA (Davis. Gastroenterology 118:S104-S114, 2000). Thus, ribavirin alone is not effective in reducing viral RNA levels. Additionally, ribavirin has significant toxicity and is known to induce anemia.
Treatment of HCV Infection with Interferon
Interferons (IFNs) are compounds which have been commercially available for the treatment of chronic hepatitis for nearly a decade. IFNs are glycoproteins produced by immune cells in response to viral infection. IFNs inhibit viral replication of many viruses, including HCV, and when used as the sole treatment for hepatitis C infection, IFN suppresses serum HCV-RNA to undetectable levels. Additionally, IFN normalizes serum amino transferase levels. Unfortunately, the effects of IFN are temporary and a sustained response occurs in only 8%-9% of patients chronically infected with HCV (Davis. Gastroenterology 118:S104-S114, 2000). A recent study, however, has indicated that early and prompt treatment with an interferon analogue, interferon alpha-2b of patients acutely infected with HCV can prevent chronic infection in 98% of the patients (Jaeckel et al, N.Engl. J. Med., Nov. 15, 2001, 345).
A number of patents are directed to the use of interferons to treat HCV infection. For example, U.S. Pat. No. 5,980,884 to Blatt et al. discloses methods for treatment of patients afflicted with HCV using consensus interferons.
U.S. Pat. No. 5,942,223 to Bazer et al. discloses an anti-HCV therapy using ovine or bovine interferon-tau.
U.S. Pat. No. 5,928,636 to Alber et al. discloses the combination therapy of interleukin-12 and interferon alpha for the treatment of infectious diseases including HCV.
U.S. Pat. No. 5,908,621 to Glue et al. discloses the use of polyethylene glycol modified interferon for the treatment of HCV.
U.S. Pat. No. 5,849,696 to Chretien et al. discloses the use of thymosins, alone or in combination with interferon, for treating HCV.
U.S. Pat. No. 5,830,455 to Valtuena et al. discloses a combination HCV therapy employing interferon and a free radical scavenger.
U.S. Pat. No. 5,738,845 to Imakawa discloses the use of human interferon tau proteins for treating HCV. Other interferon-based treatments for HCV are disclosed in U.S. Pat. No. 5,676,942 to Testa et al., and U.S. Pat. No. 5,372,808 to Blatt et al.
Combination of Interferon and Ribavirin
The combination of IFN and ribavirin for the treatment of HCV infection has been reported to be effective in the treatment of IFN naïve patients. (Battaglia, A. M. et al., Ann. Pharmacother. 34:487-494, 2000). Results are promising for this combination treatment both before hepatitis develops or when histological disease is present (Berenguer, M. et al. Antivir. Ther. 3(Suppl. 3):125-136, 1998). Side effects of combination therapy include hemolysis, flulike symptoms, anemia, and fatigue. (Davis. Gastroenterology 118:S104-S114, 2000).
Additional Treatments for HCV Infections
U.S. Pat. No. 5,891,874 discloses a series of benzimidazole compounds and a method for inhibiting Flaviviridae including hepatitis C and bovine diarrheal virus using such compounds.
U.S. Pat. No. 6,056,961 discloses extracts of the plant Hypericum perforatum and pharmaceutical compositions thereof for the treatment of HCV infection. Other U.S. patents disclosing plant extracts for the treatment of HCV infection include: U.S. Pat. No. 5,837,257 to Tsai et al., U.S. Pat. No. 5,725,859 to Omer et al.
U.S. Pat. No. 6,001,799 discloses a method of treating hepatitis C in non-responders to interferon comprising administering at least one thymosin.
U.S. Pat. No. 5,922,757 discloses methods for the treatment of hepatitis C involving the administration of vitamin E and other compounds with antioxidant properties.
Several patents disclose protease inhibitors for the treatment of HCV. For example, U.S. Pat. No. 6,004,933 to Spruce et al. discloses a class of cysteine protease inhibitors for inhibiting HCV.
U.S. Pat. No. 5,990,276 to Zhang et al. discloses synthetic inhibitors of hepatitis C virus NS3 protease. The inhibitor is a subsequence of a substrate of the NS3 protease or a substrate of the NS4A cofactor.
U.S. Pat. No. 5,972,347 to Eder et al. and U.S. Pat. No. 5,969,109 to Bona et al. disclose a vaccine for treating HCV.
U.S. Pat. No. 6,034,134 to Gold et al. discloses certain NMDA receptor agonists having immunodulatory, antimalarial, anti-Boma virus, and anti-HCV activities. The disclosed NMDA receptor agonists belong to a family of 1-amino-alkylcyclohexanes.
U.S. Pat. No. 6,030,960 to Morris-Natschke et al. discloses the use of certain alkyl lipids to inhibit the production of hepatitis-induced antigens, including those produced by the HCV virus.
U.S. Pat. No. 5,858,389 to Elsherbi et al. discloses the use of squalene for treating hepatitis C.
U.S. Pat. No. 5,849,800 to Smith et al. discloses the use of amantadine for treatment of Hepatitis C.
U.S. Pat. No. 5,846,964 to Ozeki et al. discloses the use of bile acids for treating HCV.
U.S. Pat. No. 5,491,135 to Blough et al. discloses the use of N-(phosphonoacetyl)-L-aspartic acid to treat flaviviruses such as HCV.
U.S. Pat. No. 5,922,857 to Han et al. disclose nucleic acids corresponding to the sequence of the pestivirus homology box IV area for controlling the translation of HCV.
The use of ribozymes to treat HCV is disclosed in U.S. Pat. No. 6,043,077 to Barber et al., and U.S. Pat. Nos. 5,869,253 and 5,610,054 to Draper et al.
PCT application WO 99/29350 discloses compositions and methods of treatment for hepatitis C infection comprising the administration of antisense oligonucleotides which are complementary and hybridizable to HCV-RNA.
U.S. Pat. No. 6,001,990 discloses antisense oligonucleotides and methods of using these antisense oligonucleotides for inhibiting HCV-RNA translation.
U.S. Pat. No. 6,027,729 discloses and claims polypeptides encoded by the HCV genome.
U.S. Pat. No. 5,128,458 discloses β-D-2′,3′-dideoxy-4′-thioribonucleosides as antiviral agents. U.S. Pat. No. 5,446,029 discloses that 2′,3′-dideoxy-3′-fluoronucleosides have anti-hepatitis activity.
Because of the serious effect of HCV infection on a host and the widespread infection of HCV, there exists a critical need for new animal and cellular models to study the pathogenesis of Flaviviridae infections, including flavivirus, pestivirus and hepacivirus infections, particularly HCV. Moreover, there is a strong need for new compounds and methods of treating Flavivirus infections including HCV.